Chapter 26:Chapter 26:Biomolecules: Amino Acids, Biomolecules: Amino Acids,

Peptides, and ProteinsPeptides, and Proteins

Why this Chapter?Why this Chapter?Amino acids are the fundamental building blocks of proteins

To see how amino acids are incorporated into proteins and the structures of proteins

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Proteins – Amides from Amino AcidsProteins – Amides from Amino AcidsAmino acids contain a basic amino group and an

acidic carboxyl group Joined as amides between the NH2 of one amino

acid and the CO2H to the next amino acidChains with fewer than 50 units are called peptidesProtein: large chains that have structural or

catalytic functions in biology

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26.1 Structures of Amino Acids26.1 Structures of Amino Acids

In neutral solution, the COOH is ionized and the NH2 is protonated

The resulting structures have “+” and “-” charges (a dipolar ion, or zwitterion)

They are like ionic salts in solution

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The Common Amino AcidsThe Common Amino Acids 20 amino acids form amides in proteins All are -amino acids - the amino and carboxyl are connected

to the same C They differ by the other substituent attached to the carbon,

called the side chain, with H as the fourth substituent Proline is a five-membered secondary amine, with N and the

C part of a five-membered ring See table 26.1 to examine names, abbreviations, physical

properties, and structures of 20 commonly occurring amino acids

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Abbreviations and CodesAbbreviations and Codes

Alanine A, Ala

Arginine R, Arg

Asparagine N, Asn

Aspartic acid D, Asp

Cysteine C, Cys

Glutamine Q, Gln

Glutamic Acid E, Glu

Glycine G, Gly

Histidine H, His

Isoleucine I, Ile

Leucine L, LeuLysine K, LysMethionine M, MetPhenylalanine F, PheProline P, ProSerine S, SerThreonine T, ThrTryptophan W, TrpTyrosine Y, TyrValine V, Val

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Chirality of Amino AcidsChirality of Amino AcidsGlycine, 2-amino-acetic acid, is achiral In all the others, the carbons of the amino

acids are centers of chiralityThe stereochemical reference for amino acids

is the Fischer projection of L-serineProteins are derived exclusively from L-amino

acids

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Types of side chainsTypes of side chainsNeutral: Fifteen of the twenty have neutral

side chainsAsp and Glu have a second COOH and are

acidicLys, Arg, His have additional basic amino

groups side chains (the N in tryptophan is a very weak base)

Cys, Ser, Tyr (OH and SH) are weak acids that are good nucleophiles

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HistidineHistidineContains an imidazole ring that is partially

protonated in neutral solutionOnly the pyridine-like, doubly bonded nitrogen

in histidine is basic. The pyrrole-like singly bonded nitrogen is nonbasic because its lone pair of electrons is part of the 6 electron aromatic imidazole ring.

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Essential Amino AcidsEssential Amino AcidsAll 20 of the amino acids are necessary for

protein synthesisHumans can synthesize only 10 of the 20The other 10 must be obtained from food

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26.2 Amino Acids, the Henderson 26.2 Amino Acids, the Henderson Hasselbalch Equation, and Isoelectric PointsHasselbalch Equation, and Isoelectric Points

In acidic solution, the carboxylate and amine are in their conjugate acid forms, an overall cation

In basic solution, the groups are in their base forms, an overall anion

In neutral solution cation and anion forms are present

This pH where the overall charge is 0 is the isoelectric point, pI

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Titration Curves of Amino AcidsTitration Curves of Amino Acids If pKa values for an amino acid are known the

fractions of each protonation state can be calculated (Henderson-Hasselbach Equation)

pH = pKa – log [A-]/[HA]This permits a titration curve to be calculated or

pKa to be determined from a titration curve

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pI Depends on Side ChainpI Depends on Side Chain

The 15 amino acids with thiol, hydroxyl groups or pure hydrocarbon side chains have pI = 5.0 to 6.5 (average of the pKa’s)

D and E have acidic side chains and a lower pIH, R, K have basic side chains and higher pI

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ElectrophoresisElectrophoresisProteins have an overall pI that depends on the

net acidity/basicity of the side chainsThe differences in pI can be used for separating

proteins on a solid phase permeated with liquidDifferent amino acids migrate at different rates,

depending on their isoelectric points and on the pH of the aqueous buffer

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26.3 Synthesis of Amino 26.3 Synthesis of Amino AcidsAcids

Bromination of a carboxylic acid by treatment with Br2 and PBr3 (22.4) then use NH3 or phthalimide to displace Br

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The Amidomalonate SynthesisThe Amidomalonate SynthesisBased on malonic ester synthesisConvert diethyl acetamidomalonate into enolate

ion with base, followed by alkylation with a primary alkyl halide

Hydrolysis of the amide protecting group and the esters and decarboxylation yields an -amino

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Reductive Amination of Reductive Amination of -Keto Acids-Keto Acids

Reaction of an -keto acid with NH3 and a reducing agent (see Section 24.6) produces an -amino acid

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Enantioselective Synthesis of Enantioselective Synthesis of Amino AcidsAmino Acids

Amino acids (except glycine) are chiral and pure enantiomers are required for any protein or peptide synthesis

Resolution of racemic mixtures is inherently ineffecient since at least half the material is discarded

An efficient alternative is enantioselective synthesis

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Enantioselective Synthesis of Enantioselective Synthesis of Amino Acids (cont’d)Amino Acids (cont’d)

Chiral reaction catalyst creates diastereomeric transition states that lead to an excess of one enantiomeric product

Hydrogenation of a Z enamido acid with a chiral hydrogenation catalyst produces S enantiomer selectively

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26.4 Peptides and Proteins26.4 Peptides and ProteinsProteins and peptides are amino acid polymers in

which the individual amino acid units, called residues, are linked together by amide bonds, or peptide bonds

An amino group from one residue forms an amide bond with the carboxyl of a second residue

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Peptide LinkagesPeptide Linkages Two dipeptides can result from reaction between A and S,

depending on which COOH reacts with which NH2 we get AS or SA

The long, repetitive sequence of NCHCO atoms that make up a continuous chain is called the protein’s backbone

Peptides are always written with the N-terminal amino acid (the one with the free NH2 group) on the left and the C-terminal amino acid (the one with the free CO2H group) on the right

Alanylserine is abbreviated Ala-Ser (or A-S), and serylalanine is abbreviated Ser-Ala (or S-A)

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26.5 Amino Acid Analysis of Peptides26.5 Amino Acid Analysis of PeptidesThe sequence of amino acids in a pure protein

is specified genetically If a protein is isolated it can be analyzed for its

sequenceThe composition of amino acids can be obtained

by automated chromatography and quantitative measurement of eluted materials using a reaction with ninhydrin that produces an intense purple color

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Amino Acid Analysis ChromatogramAmino Acid Analysis Chromatogram

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26.6 Peptide Sequencing: The 26.6 Peptide Sequencing: The Edman DegradationEdman Degradation

The Edman degradation cleaves amino acids one at a time from the N-terminus and forms a detectable, separable derivative for each amino acid

Examine Figure 26.4

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26.7 Peptide Synthesis26.7 Peptide SynthesisPeptide synthesis requires that different amide

bonds must be formed in a desired sequenceThe growing chain is protected at the carboxyl

terminal and added amino acids are N-protectedAfter peptide bond formation, N-protection is

removed

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Carboxyl Protecting GroupsCarboxyl Protecting GroupsUsually converted into methyl or benzyl estersRemoved by mild hydrolysis with aqueous NaOHBenzyl esters are cleaved by catalytic

hydrogenolysis of the weak benzylic C–O bond

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Amino Group ProtectionAmino Group ProtectionAn amide that is less stable than the protein

amide is formed and then removedThe tert-butoxycarbonyl amide (BOC) protecting

group is introduced with di-tert-butyl dicarbonateRemoved by brief treatment with trifluoroacetic

acid

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Peptide CouplingPeptide CouplingAmides are formed by treating a mixture of an

acid and amine with dicyclohexylcarbodiimide (DCC)

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Overall Steps in Peptide SynthesisOverall Steps in Peptide Synthesis

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26.8 Automated Peptide Synthesis: The 26.8 Automated Peptide Synthesis: The Merrifield Solid-Phase TechniqueMerrifield Solid-Phase Technique

Peptides are connected to beads of polystyrene, reacted, cycled and cleaved at the end

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26.9 Protein Structure26.9 Protein StructureThe primary structure of a protein is simply

the amino acid sequence.The secondary structure of a protein

describes how segments of the peptide backbone orient into a regular pattern.

The tertiary structure describes how the entire protein molecule coils into an overall three-dimensional shape.

The quaternary structure describes how different protein molecules come together to yield large aggregate structures

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--HelixHelix-helix stabilized by H-bonds between amide

N–H groups and C=O groups four residues away -helical segments in their chains

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--Pleated SheetPleated Sheet -pleated sheet secondary structure is exhibited by

polypeptide chains lined up in a parallel arrangement, and held together by hydrogen bonds between chains

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Denaturation of ProteinsDenaturation of Proteins The tertiary structure of a globular protein is the result

of many intramolecular attractions that can be disrupted by a change of the environment, causing the protein to become denatured

Solubility is drastically decreased as in heating egg white, where the albumins unfold and coagulate

Enzymes also lose all catalytic activity when denatured

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26.10 Enzymes and Coenzymes26.10 Enzymes and Coenzymes

An enzyme is a protein that acts as a catalyst for a biological reaction.

Most enzymes are specific for substrates while enzymes involved in digestion, such as papin attack many substrates

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Types of Enzymes by FunctionTypes of Enzymes by FunctionEnzymes are usually grouped according to the kind

of reaction they catalyze, not by their structures

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26.11 How Do Enzymes Work? 26.11 How Do Enzymes Work? Citrate SynthaseCitrate Synthase

Citrate synthase catalyzes a mixed Claisen condensation of acetyl CoA and oxaloacetate to give citrate

Normally Claisen condensations require a strong base in an alcohol solvent but citrate synthetase operates in neutral solution

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The Structure of Citrate SynthaseThe Structure of Citrate Synthase

Determined by X-ray crystallography

Enzyme is very large compared to substrates, creating a complete environment for the reaction

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Mechanism of Citrate SynthetaseMechanism of Citrate Synthetase

A cleft with functional groups binds oxaloacetateAnother cleft opens for acetyl CoA with H 274

and D 375, whose carboxylate abstracts a proton from acetyl CoA

The enolate (stabilized by a cation) adds to the carbonyl group of oxaloacetate

The thiol ester in citryl CoA is hydrolyzed

An amino acid would have the following An amino acid would have the following structure under which of the following structure under which of the following conditions?conditions?

1 2 3 4 5

20% 20% 20%20%20%

1. neutral solution2. acid solution3. basic solution4. only at pH 6.55. under no

conditions

All of the following amino acids All of the following amino acids are primary amines except:are primary amines except:

1 2 3 4 5

20% 20% 20%20%20%

1. proline2. thyroxine3. glutamic acid4. lysine5. glycine

The amino acid shown below is The amino acid shown below is which of the following:which of the following:

1 2 3 4 5

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1. histidine2. lysine3. aspartic acid4. glycine5. none of these

In the structure of histidine,In the structure of histidine,

1 2 3 4 5

20% 20% 20%20%20%1. the side chain is acidic

2. both of the nitrogens in the ring structure are basic

3. one nitrogen in the ring structure is acidic and one is basic

4. there is one basic nitrogen in the imidazole ring

5. there is no carboxyl group

One can always calculate the pI of an One can always calculate the pI of an amino acid byamino acid by

1 2 3 4 5

20% 20% 20%20%20%

1. averaging the pKa of the α-carboxyl group and the α-amino group

2. averaging two pKa values, but which ones are averaged will depend on which amino acid it is

3. averaging the pKa of the α-carboxyl and the side chain

4. averaging the pKa of the α-amino and the side chain

5. averaging the pKa values of the α-amino, α-carboxyl, and side chain

The reductive amination of the structure The reductive amination of the structure shown below would give which amino shown below would give which amino acid product?acid product?

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1. aspartic acid2. glutamic acid3. cysteine4. alanine5. none of these

In a peptide abbreviated G-G-A-S-In a peptide abbreviated G-G-A-S-Q-K-K,Q-K-K,

1 2 3 4 5

20% 20% 20%20%20%1. glutamic acid is the N-terminus

2. lysine is the C-terminus

3. there are 7 residues

4. all of these5. both lysine is the

C-terminus and there are 7 residues

In a peptide bond, there is In a peptide bond, there is restricted rotation between which of restricted rotation between which of the following atoms?the following atoms?

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1. the nitrogen and the α-carbon

2. the nitrogen and the carboxyl carbon

3. the carboxyl carbon and the α-carbon

4. the α-carbon and the side chain

5. none of these

What part of an amino acid ends up What part of an amino acid ends up bound to the ninhydrin molecule in the bound to the ninhydrin molecule in the final purple colored product of a final purple colored product of a ninhydrin reaction?ninhydrin reaction?

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1. the nitrogen from the α-amino group

2. the nitrogen from any side chain

3. the carbon from the α-carboxyl group

4. the α-carbon5. the hydrogen bound

to the α-carbon

Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-HisSer-Arg-His

1 2 3 4 5

20% 20% 20%20%20%

1. 12. 23. 34. 45. 5

Chymotrypsin would cleave the above peptide into how many fragments?

Which of the following is out of order for the Which of the following is out of order for the Merrifield solid-phase method of peptide Merrifield solid-phase method of peptide synthesis?synthesis?

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20% 20% 20%20%20%1. An amino acid is

protected as a BOC-derivative

2. The BOC-protected amino acid is linked to the polystyrene polymer

3. The polymer-bound amino acid is treated with trifluoroacetic acid to remove the BOC group

4. A second BOC-protected amino acid is coupled to the first by reaction with DCC

5. All of the steps are in the correct order

The α-helix is an example of The α-helix is an example of what level of protein structure?what level of protein structure?

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20% 20% 20%20%20%

1. primary structure2. secondary

structure3. tertiary structure4. quaternary

structure5. both primary

structure and secondary structure

The structure shown below is:The structure shown below is:

1. an α-helix2. a β-pleated sheet3. myoglobin4. concavalin A5. a denatured

protein 1 2 3 4 5

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Enzymes typically bind most Enzymes typically bind most tightly to which structure?tightly to which structure?

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1. the substrates2. the products3. the transition

state4. a coenzyme5. the enzymes bind

to all of these equally well

An enzyme that introduces a An enzyme that introduces a double bond would be called double bond would be called a(n):a(n):

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1. oxidoreductase2. transferase3. hydrolase4. lyase5. isomerase

Which of the following amino Which of the following amino acids is necessary for disulfide acids is necessary for disulfide bonds?bonds?

1 2 3 4 5

20% 20% 20%20%20%

1. cysteine2. histidine3. serine4. methionine5. tyrosine

Which term describes the Which term describes the breakage of a peptide bond?breakage of a peptide bond?

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20% 20% 20%20%20%

1. α-cleavage2. β-elimination3. hydrolysis4. condensation5. chain

termination